Microarray-based sample analysis system

ABSTRACT

A microarray-based sample analysis (MBSA) system includes a cartridge holder adapted to receive a replaceable cartridge that is configured to receive a detachable, replaceable sample analysis unit containing one or more reaction chambers for sample analysis; a fluid control subsystem that controls fluid flow; and an optical subsystem configured to capture an image of the microarray.

RELEVANT APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/886,201, filed on Sep. 20, 2010, which claims priority of U.S.Provisional Application No. 61/272,397, filed on Sep. 21, 2009. Theentirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

The technical field is biotechnology and, more specifically, methods andapparatus for analysis of biomolecules.

BACKGROUND

It is desirable to have an analytical instrument that possesses bothsample preparation and sample analysis functions. It is also desirableto have an analytical instrument that is light and small and can beproduced at a low cost. However, microfluidic challenges have impededthe development of such analytical devices. These challenges are due, inpart, to the fluid dynamics at small scales. For example, as thediameter of a microfluidic channel decreases, the pressure drop acrossthe channel increases by the 4^(th) power, according to theHagen-Poiseuille equation. When employing complex microfluidicgeometries, these large pressure drops can result in flow patterns thatare very difficult to predict, particularly with air bubbles in thesystem. The thermal expansion of air is more than five times greaterthan liquid, causing additional challenges.

SUMMARY

An integrated cartridge for sample processing and analysis is disclosed.The integrated cartridge contains a sample preparation chamber having asample inlet and a sample outlet, and a sample purification chamberadapted to receive a replaceable sample purification unit containing ahousing and an extraction filter inside the housing. The extractionfilter specifically binds to a molecule of interest. The samplepurification chamber has a sample inlet that is in fluid communicationwith the sample outlet of the sample preparation chamber.

Also disclosed is a microarray-based sample analysis (MBSA) system. TheMBSA system includes a cartridge holder adapted to receive a detachablecartridge that is configured to receive a detachable, replaceable sampleanalysis unit having a reaction chamber and a microarray, a fluidcontrol subsystem that controls fluid flow, and an optical subsystemconfigured to capture an image of the microarray.

BRIEF DESCRIPTION OF DRAWINGS

The detailed description will refer to the following drawings in which:

FIGS. 1A and 1B are schematic drawings showing embodiments of theintegrated cartridge.

FIG. 2 is a schematic drawing showing an integrated cartridge with celllysis means.

FIG. 3A-3C are schematics showing different embodiments of the proteinpurification components of a dual-function integrated cartridge.

FIG. 4 is a block diagram of a microarray-based sample analysis (MBSA)system.

FIG. 5 is a schematic of the fluidic subsystem showing pumps andselection valves (SV) connected to the integrated cartridge.

FIG. 6A is a schematic showing an integrated cartridge residing in afluidic manifold without the thermocycler bladders.

FIG. 6B is a schematic showing an integrated cartridge residing in afluidic manifold with the thermocycler bladders.

FIG. 7 is a schematic showing the location of the on-board microfluidiccartridge valves in an integrated cartridge.

FIG. 8 is a schematic showing an embodiment of an on-board microfluidiccartridge valve.

FIG. 9 is a schematic showing an embodiment of the chamber arrangementwithin a flow cell.

FIG. 10 is a diagram showing PCR/APEX allele signal ratio resultsobtained for an eye color SNP at position RS1800407. PCR was performedin a Akonni flow cell positioned in a bladder thermocycler, in 0.2 mlPCR tube positioned in a MJ thermal cycler, and in Cepheid tubepositioned in the bladder thermocycler. APEX was performed offline forone hour. The results indicate comparable APEX signals for all three PCRapproaches.

FIG. 11A is a schematic of an embodiment of the optical subsystem with alaser light source.

FIG. 11B shows a typical image of a Cy3 array with a pitch of 300 μmtaken with the optical subsystem described in FIG. 11A with the co-axialillumination.

FIG. 12A schematic of another embodiment of the optical subsystem with alaser light source.

FIG. 12B shows a typical image of am 11×18 Cy3 array taken with theoptical subsystem of FIG. 12A.

FIG. 13A is a schematic showing an embodiment of the optical subsystemwith a high-brightness LED.

FIGS. 13B is a schematic showing an optical train for fluorescence andFII imaging.

FIGS. 13C is a schematic showing another optical train for fluorescenceand FII imaging.

FIGS. 13D is a schematic showing a collimated source used in FIGS. 13Band 13C.

FIG. 14A is an FII image of a gel array.

FIG. 14B is a diagram showing the normalized pixel intensity profile ofan array element in FIG. 14A.

FIG. 15 is a composite of images showing a gel array image processed bydifferent methods available in the ImageJ software. Panel A, originalimage; Panel B, ImageJ processing: Process=>Find Edges; Panel C, ImageJprocessing: Process=>Filters=>Median, R=5; Panel D, ImageJ processing:Process=>Filters=>Mean, R=5; Panel E, ImageJ processing: Image=>AdjustThreshold; Panel F, ImageJ processing: Process=>Filters=>Maximum . . .R=2; Panel G, ImageJ processing: Process=>Binary=>Find Maxima; Panel H,Superposition of the images showing respectively spot centers found andspot boundaries detected.

FIG. 16 is a composite showing evaluation of spot morphology. Panel A:image obtained in the fluorescence imaging mode; Panel B: FII image ofthe same array obtained using oblique illumination with collimated beam;Panel C: Image processing in ImageJ using Process=>Find Edges; Panel D:Image processing in ImageJ using Process=>Filters=>Median, R=2;Image=>Adjust threshold.

FIGS. 17A and 17B are graphs showing evaluation of morphology for thegel spots in panel A of FIG. 15

FIG. 18A is picture showing detection of S. pyogenes with a prototypeMBSA system and an Aurora imager.

FIG. 18B is picture showing detection of S. pyogenes with a prototypeMBSA system and an Akonni imager.

FIG. 19A is a picture showing detection of B. anthraces with a prototypeMBSA system and the imaging approach shown in FIG. 11A.

FIG. 19B is an array map of the microarray used in FIG. 19A.

FIG. 20 is diagram showing PCR results of S. pyogenes DNA extractedusing an integrated cartridge with a bead mixer. Mixer 1 and mixer 2: S.pyogenes DNA extracted in two separated experiment using an integratedcartridge with a bead mixer. Vortex: S. pyogenes DNA extracted usingstandard vortexing method. NTC: negative control.

FIG. 21A shows the microarray results for 6-plex PCR products. The upperleft panel is a microarray image with arrows pointing to the six PCRproduct generated in the multiplex PCR. The upper right panel is themicroarray map. The lower panel shows probe number/primeridentifier/fluorescent signal intensities for PCR product generatedunder three different annealing temperatures.

FIG. 21B is a diagram showing real-time PCR analysis ofcartridge-purified blood DNA.

FIG. 22 shows thermocycling profile with PID pump and heater control.Line A shows the temperature of the hot zone, line B shows the coldzone, line C shows the temperature of a thermocouple sandwiched betweenthe bladders, and line D shows the temperature of the working fluid justprior to entering the bladder.

FIG. 23 is a drawing showing an embodiment of a dual-function integratedcartridge.

FIG. 24 is a schematic showing an embodiment of a complete MBSA system.

DETAILED DESCRIPTION

This description is intended to be read in connection with theaccompanying drawings, which are to be considered part of the entirewritten description of this invention. The drawing figures are notnecessarily to scale and certain features of the invention may be shownexaggerated in scale or in somewhat schematic form in the interest ofclarity and conciseness. In the description, relative terms such as“front,” “back,” “up,” “down,” “top” and “bottom,” as well asderivatives thereof, should be construed to refer to the orientation asthen described or as shown in the drawing figure under discussion. Theserelative terms are for convenience of description and normally are notintended to require a particular orientation. Terms concerningattachments, coupling and the like, such as “connected” and “attached,”refer to a relationship wherein structures are secured or attached toone another either directly or indirectly through interveningstructures, as well as both movable or rigid attachments orrelationships, unless expressly described otherwise.

In describing preferred embodiments of the present invention, specificterminology is employed for the sake of clarity. However, the inventionis not intended to be limited to the specific terminology so selected.It is to be understood that each specific element includes all technicalequivalents which operate in a similar manner to accomplish a similarpurpose.

An integrated cartridge for sample processing and analysis is disclosed.The integrated cartridge includes a sample preparation chamber, a samplepurification chamber and a detachable sample analysis unit. The samplepreparation chamber has a sample inlet and sample outlet and is in fluidcommunication with the sample purification chamber. The samplepurification chamber contains an extraction filter that specificallybinds to a molecule of interest. The detachable sample analysis unitincludes at least one sample analysis chamber that contains amicroarray.

FIG. 1A shows an embodiment of an integrated cartridge. The integratedcartridge allows for the extraction of biomolecules, such aspolypeptides and polynucleotides, and subsequent analysis of thebiomolecules within the same cartridge. In this embodiment, theintegrated cartridge 100 contains a cartridge body 1 and a sampleanalysis unit 2. The cartridge body 1 contains a sample preparationchamber 10, a sample purification chamber 20 in fluid communication withthe sample preparation chamber 10, an sample elution chamber 30 in fluidcommunication with the sample purification chamber 20, a waste chamber40 in fluid communication with the sample purification chamber 20, aproduct waste chamber 50 in fluid communication with the sample analysisunit 2 when it is attached to the cartridge body 1, fluidic channels 60that connect the chambers to each other, and a fluidic interface 70 thatallows the integrated cartridge 100 to be connected to a cartridge base(not shown). In one embodiment, the sample analysis unit 2 is anintegrated part of the cartridge body 1. In another embodiment, thesample analysis unit 2 is detachable from the cartridge body 1. FIG. 1Bis a schematic drawing showing the same cartridge 100 with on-boardvalves 80 that control fluid flow in each chamber.

The sample preparation chamber 10 has a sample inlet 11 at an upperportion of the chamber and a sample outlet 12 at a lower portion of thechamber. In the embodiments shown in FIGS. 1A and 1B, the sample inlet11 is located at the top of the sample preparation chamber 10 and thesample outlet 12 is located at the bottom of the sample preparationchamber 10. In one embodiment, the sample inlet 11 is a dome valve. Whenconnected to a pump, the sample outlet 12 may also serve as an airinlet. Sample mixing inside the sample preparation chamber may beachieved by pumping a gas, such as air or an inert gas (e.g., nitrogen),into the sample preparation chamber 10 from the sample outlet 12. Thegas forms bubbles that migrate from the bottom to the top of the chamber10 and mix the sample during the process.

In another embodiment, the gas is heated to provide a means of unifoiiily heating the sample/reagent mixture in the sample preparation chamber10.

The sample purification chamber 20 contains an extraction filter 21 thatbinds specifically to an analyte. Examples of analytes include, but arenot limited to, polynucleotides, polypeptides, lipids, andpolysaccharides. In one embodiment, the extraction filter is a silicaextraction filter that specifically binds to DNA. In another embodimentthe extraction filter is a porous material that specifically binds to aprotein. In one embodiment, the extraction filter 21 is located in aremovable sample purification unit 22 that can be easily detached fromthe cartridge body 1 and replaced with a new sample purification unit22. The sample purification unit 22 comprises a housing with an inletand an outlet, and an extraction filter 21 that specifically binds to ananalyte. In one embodiment, the sample purification unit 22 is in theform of a pipet tip. In a preferred embodiment, the pipet tip is sizedto fit within the confines of the sample purification chamber 20. Inanother preferred embodiment, the extraction filter 21 is the glass fritdescribed in U.S. patent application Ser. Nos. 11/933,113 and12/213,942, both of which are herein incorporated by reference in theirentirety.

The elution chamber 30 is connected to both the sample purificationchamber 20 and sample analysis unit 2 when it is attached to thecartridge body 1. In certain embodiments, the elution chamber 30 iseliminated from the integrated cartridge and the sample purificationchamber 20 is connected directly to the sample analysis unit 2.

The integrated cartridge 100 is designed to operate in the uprightposition as shown in FIGS. 1A and 1B. The sample preparation chamber andsample purification chamber are designed to have a diameter that issufficient to allow bubbles to rise to the top.

The sample analysis unit 2 may contain one or more chambers for sampleanalysis. In one embodiment, the sample analysis unit 2 contains amicroarray chamber 3 that contains a microarray 4 for the analysis ofthe analyte. The microarray 4 can be a microarray of any type. In oneembodiment, the microarray 4 is a DNA array. In another embodiment, themicroarray 4 is a protein or peptide array. In another embodiment, themicroarray is a gel element array described in e.g., U.S. Pat. Nos.5,741,700, 5,770,721; 5,981,734, and 6,656,725, and U.S. patentapplication Ser. Nos. 10/068,474, 11/425,667 and 60/793,176, which arehereby incorporated by reference in their entirety. In yet anotherembodiment, the microarray 4 is an antibody array.

In one embodiment, the microarray chamber 3 also serves as a reactionchamber for an amplification reaction, such as polymerase chain reaction(PCR) or arrayed-primer extension (APEX).

In another embodiment, the sample analysis unit 2 further contains anamplification chamber 5. In one embodiment, the amplification chamber 5is a PCR chamber that can be heated and cooled repetitively to amplify aDNA target inside the amplification chamber 5.

The sample analysis unit 2 and/or the cartridge body 1, are made frommaterials capable of withstanding thermocycling, and immune to solventssuch as ethanol. The cartridge body can be machined or injection molded.

In one embodiment, the microarray chamber 3 and/or the amplificationchamber 5 have a hydrophilic interior surface. In a preferredembodiment, the microarray chamber 3 and/or the amplification chamber 5are hydrophilic flow cells described in U.S. patent application Ser. No.12/149,865, which is incorporated herein by reference in its entirety.

During operation, the cartridge 100 is inserted into a fluidic dockingstation. In one embodiment, fluidic docking station engages with thecartridge 100 through luer tapered bosses that activate luer-activatedvalves on the cartridge. A sample, such as a cell suspension, is loadedinto the sample preparation chamber 10 of the integrated cartridge 100through the sample inlet 11. A cell lysis buffer is added to the samplepreparation chamber 10, and mixed with the sample by air bubbles thatmigrate from the bottom to the top of the sample preparation chamber 10.The air bubbles are controlled by regulated air flow from an air pump.This air may be heated for protocols that require incubation at anelevated temperature. The mixed sample is then introduced into thesample purification chamber 20, which contains the extraction filter 21that specifically binds to the analyte of interest. The sample passesthrough the extraction filter 21 to allow the analyte of interest tobind to the extraction filter 21. In certain embodiments, the sample iscycled back and forth across the extraction filter 21 a number of timesto improve efficiency of analyte binding. The unbound sample is thendirected to the waste chamber 40 through on-board cartridge pin valves80 and fluidic channels 60. Leaving the waste in the cartridge preventscontamination with subsequent samples.

The extraction filter 21 is washed with a wash buffer one or more times.Used wash buffer is directed to the waste chamber 40 through on-boardcartridge valves 80 and fluidic channels 60. In one embodiment, the washbuffer is an ethanol-based wash buffer and is cycled 5 times through theextraction filter 21. An elution buffer is then introduced into thesample purification chamber 20 through the extraction filter 21. Theeluted analyte is directed into the elution chamber 30, which removesthe bubbles in the eluant. The eluted analyte is then used for thesubsequent sample analysis in the sample analysis unit. Aliquots of theeluted analyte may also be removed for other types of analysis. In oneembodiment, an eluted DNA sample is directed into the amplificationchamber 5 for PCR amplification. Temperature change in the amplificationchamber 5 may be achieved by oscillating two temperature fluids throughflexible bladders that makes intimate contact with the amplificationchamber 5, as described, for example, in described in U.S. patentapplication Ser. Nos. 11/843,843 and 12/232,669, which are incorporatedherein by reference in their entirety. The on-board cartridge valves 80switch between thermally-controlled reservoirs to provide a rapid changein temperature. The amplification product is directed into themicroarray chamber for hybridization to the microarray.

Integrated Cartridge with Cell Lysis Beads

In certain embodiments, the sample preparation chamber 10 furtherincludes sample lysis means for lysing cell samples. In an embodimentshown in FIG. 2, the sample preparation chamber 10 contains a pluralityof cell lysis beads 13 and a magnetic stirring element 14 with a magnet15. A suspension of intact cells is added to the sample preparationchamber 10 and a rotating magnetic field is applied to the samplepreparation chamber 10 to rotate the magnetic stirring element 14 at arotation speed sufficient to lyse the cells.

As used herein, the term “cell” refers to eukaryotic cells, prokaryoticcells, and components or fragments thereof. The term “cell” includesparasites, bacteria, bacteria spores, fungi, virus particles, as well asan aggregation of cells such as multi-cell organisms, tissues andfragments thereof. The term “cell suspension” refers to a mixture ofcells and a liquid medium, wherein the cells are suspended in the liquidmedium. Preferably, the cells are suspended at a concentration that isnot too thick or viscous to interfere with the movement of the magneticstir element.

In a certain embodiment, eukaryotic or prokaryotic cells are suspendedin the concentration range of 1×10² to 1×10⁵ cells/ml. In anotherembodiment, eukaryotic or prokaryotic cells are suspended in theconcentration range of 1×10³ to 1×10⁴ cells/ml. In other embodiment,virus particles are suspended in the concentration range of 1×10⁷ to1×10¹³ particles/ml. In yet other embodiment, virus particles aresuspended in the concentration range of 1×10⁹ to 1×10¹¹ particles/ml.

The liquid medium can be isotonic, hypotonic, or hypertonic. In someembodiments, the liquid medium is aqueous. In certain embodiments, theliquid medium contains a buffer and/or at least one salt or acombination of salts. In some embodiments, the pH of the liquid mediumranges from about 5 to about 8, from about 6 to 8, or from about 6.5 toabout 8.5. A variety of pH buffers may be used to achieve the desiredpH. Suitable buffers include, but are not limited to, Tris, MES,Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES,HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, Tricine, Gly-Gly,Bicine, and a phosphate buffer (e.g., sodium phosphate orsodium-potassium phosphate, among others). The liquid medium maycomprise from about 10 mM to about 100 mM buffer, about 25 mM to about75 mM buffer, or from about 40 mM to about 60 mM buffer, among others.The type and amount of the buffer used in the liquid medium can varyfrom application to application. In some embodiments, the liquid mediumhas a pH of about 7.4, which can be achieved using about 50 mM Trisbuffer. In some embodiments the liquid medium is water.

The cell lysis beads can be any particle-like or bead-like material thathas a hardness greater than the hardness of the cells to be lysed. Thecell lysis beads may be made of plastic, glass, ceramics, or any othernon-magnetic materials, such as non-magnetic metal beads. In oneembodiment, the cell lysis beads are rotationally symmetric to one axis(e.g., spherical, rounded, oval, elliptic, egg-shaped, anddroplet-shaped particles). In other embodiments, the cell lysis beadshave polyhedron shapes. In other embodiments, the cell lysis beads areirregular shaped particles. In yet other embodiments, the cell lysisbeads are particles with protrusions.

In one embodiment, the cell lysis beads have diameters in the range of10-1,000 μm. In other embodiments, the cell lysis beads have diametersin the range of 20-400 μm. In yet other embodiments, the cell lysisbeads have diameters in the range of 50-200 μm.

The magnetic stir element can be of any shape and has dimensions thatmatch the container. In other words, the magnetic stir element should besmall enough to be placed into the container and to spin or stir withinthe container. It is within the knowledge of a person of ordinary skillin the art to choose a magnetic stir element of appropriate sizes for agiven container. The magnetic stir eement can be a bar-shaped,cylinder-shaped, rod-shaped, cross-shaped, V-shaped, triangular,rectangular or disc-shaped stir element. In one embodiment, the magneticstirring element has a rectangular shape. In another embodiment, themagnetic stirrer has a two-pronged tuning fork shape. In yet anotherembodiment, the magnetic stirrer has a V-like shape. In certainembodiments, the magnetic stir element is coated with a chemically inertmaterial, such as plastics, glass or porcelain.

In one embodiment, the cell lysis beads 13 and/or the magnetic stirringelement 14 are pre-packed into the sample preparation chamber 10. Inanother embodiment, the cell lysis beads 13 and/or the magnetic stirringelement 14 are pre-packed into a removable sample lysis unit that can beeasily placed into the sample preparation chamber 10 and discarded aftercell lysis. In yet another embodiment, the cell lysis beads 13 and/orthe magnetic stirring element 14 are added to the sample preparationchamber 10 with the cell suspension. The cell lysis beads 13, themagnetic stirring element 14 and the cell suspension may be placed intothe sample preparation chamber 10 in any order. In one embodiment, thecell suspension is loaded into the sample preparation chamber 10 firstand followed with either the cell lysis beads or the magnetic stirringelement. In another embodiment, the hard beads and/or the magneticstirring element are placed into the sample preparation chamber 10first, followed with the cell suspension.

The cartridge is then placed in close proximity to a magnetic stirrer.The cell suspension is stirred with the magnetic stirring element at arotation speed sufficient to lyse the cells inside the container. Theoptimal stirring speed and duration can be empirically determined basedon the cells, viruses or tissues to be lysed. The appropriate rotationspeed is application dependent and can be determined by a person ofordinary skill in the art. Generally speaking, the rotational speedsufficient to lyse the cells is determined by factors such as the typeof cells, the concentration of cell suspension, the size and shape ofthe magnetic stirring element, the amount, size, shape and hardness ofthe hard beads, and the size, shape and interior surface roughness ofthe container. In certain embodiments, the container in the shape of atest tube or Eppendorf tube is placed in a rack on a standard laboratorymagnetic stirrer plate and is stirred at the highest speed setting.

In certain embodiments, lysing of particular cell types can befacilitated by adding additives to the cell suspension prior to thestirring step. Examples of additives include enzymes, detergents,surfactants and other chemicals such as bases and acids. It has beenfound that alkaline conditions (e.g. 10 mM NaOH) may enhance the lysisefficiency for certain types of cells. It should be noted, however, thatthe additive should not interfere with the downstream reactions in thesample analysis process. The cell suspension may also be heated duringstirring to enhance the lysis efficiency.

Other embodiments of the bead/magnetic stirrer lysis methods and systemsare described in U.S. Provisional Application No. 61/272,396, which isincorporated herein by reference in its entirety.

Besides the bead/magnetic stirrer method, cells in the samplepreparation chamber 10 may be lysed with other methods such as beadbeating, vortexing, sonication and chemical lysis.

To avoid problems commonly associated with microfluidic devices, theintegrated cartridge may contain one or more of the following features(1) The microfluidic channels in the integrated cartridge aresufficiently large (0.2-1 mm, preferably 0.5 mm in diameter) to reducebackpressure and enable reproducible injection molded features, whichcan be highly variable when creating microfluidic geometries below 0.1mm. (2) The interior surface of the microfluidic channels are fullycovered, or partially covered or coated with a hydrophilic film toreduce bubble trapping inside the microfluidic channels. (3) Thereaction chambers (i.e., the sample preparation chamber, the samplepurification chamber and the sample elution chamber) in the integratedcartridge are designed in a vertically-oriented tower shape to ensurethat bubbles rise to the top of the chamber due to the densitydifference of air compared with water. The tower design also allows forsample mixing in the tower by flowing air into the sample from thebottom of the tower. The liquid is vigorously mixed by the air bubblesrising from the bottom to the top. (5) The extraction filter in theintegrated cartridge has a large porosity to minimize back pressure. (6)The on-board cartridge valves in the integrated cartridge shut offfluidic pathways and prevent liquids from flowing in unwanted paths. (7)The cartridge is designed so that the liquid is controlled by precisionpumps and selection valves off the disposable cartridge. The liquidmetering occurs on volumes greater than 10 μL, to prevent small airbubbles from causing large variations in reagent proportions.

Examples of the hydrophilic material that can be used for thehydrophilic cover or coating include, but are not limited to,hydrophilic polymers such as poly(N-vinyl lactams),poly(vinylpyrrolidone), poly(ethylene oxide), poly(propylene oxide),polyacrylamides, cellulosics, methyl cellulose, polyanhydrides,polyacrylic acids, polyvinyl alcohols, polyvinyl ethers, alkylphenolethoxylates, complex polyol mono-esters, polyoxyethylene esters of oleicacid, polyoxyethylene sorbitan esters of oleic acid, and sorbitan estersof fatty acids; inorganic hydrophilic materials such as inorganic oxide,gold, zeolite, and diamond-like carbon; and surfactants such as TritonX-100, Tween, Sodium dodecyl sulfate (SDS), ammonium lauryl sulfate,alkyl sulfate salts, sodium lauryl ether sulfate (SLES), alkyl benzenesulfonate, soaps, fatty acid salts, cetyl trimethylammonium bromide(CTAB) a.k.a. hexadecyl trimethyl ammonium bromide,alkyltrimethylammonium salts, cetylpyridinium chloride (CPC),polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC),benzethonium chloride (BZT), dodecyl betaine, dodecyl dimethylamineoxide, cocamidopropyl betaine, coco ampho glycinate alkyl poly(ethyleneoxide), copolymers of poly(ethylene oxide) and polypropylene oxide)(commercially called Poloxamers or Poloxamines), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, cocamide TEA. Surfactantscan be mixed with reaction polymers such as polyurethanes and epoxies toserve as a hydrophilic coating.

Simplified Integrated Cartridge

Also disclosed is a simplified cartridge for sample processing. Thecartridge includes a sample preparation chamber having an inlet and anoutlet and a sample purification chamber having an inlet and an outlet.The sample preparation chamber contains a magnetic stir element and aplurality of cell lysis beads disposed therein. The sample purificationchamber contains an extraction filter and is in fluidic communicationwith the sample preparation chamber.

In one embodiment, the magnetic stir element and the plurality of celllysis beads are pre-packed in a container that can be easily inserted orremoved from the sample preparation chamber and the extraction filter ispre-packed in a container that can be easily inserted or removed fromthe sample purification chamber.

Dual-Function Integrated Cartridge

Also disclosed is a dual-function cartridge that contains both proteinand nucleic acid purification capabilities. Briefly, the dual-functioncartridge contains a nucleic acid purification module and a proteinpurification module. Each module contains a sample purification chamberand a sample elution chamber. In one embodiment, the modules share asingle sample preparation chamber and/or a single waste chamber. Inanother embodiment, the modules share a single sample preparationchamber, but each module contains its own waste chamber. In yet anotherembodiment, each module has its own sample preparation chamber and wastechamber. Appropriate cartridge base setup can be constructed to allowsimultaneous purification of nucleic acid and protein or serialpurification of nucleic acid and protein.

In one embodiment, a sample is processed in a serial fashion. As shownin FIG. 3A, the sample is processed for nucleic acid purification first.The nucleic acid-deprived sample in the nucleic waste chamber isaspirated into a second purification chamber (e.g., a TruTip) forprotein purification and is then dispensed into a second waste chamber.The reagents are driven by a bi-directional pump such as the milliGATpump. Protein washing buffer may be added directly to the secondpurification chamber from the buffer reservoir (not shown).

In another embodiment, the integrated cartridge is modified so that thecartridge valve on the second purification chamber may serve as asyringe-type pump. As shown in FIG. 3B, only a single waste chamber isneeded. Specifically, the cartridge is modified to have a reservoirpocket connected to the cartridge valve on the second purificationchamber (shown as the “syringe pump” in FIG. 3B), so that liquid in thenucleic acid waste chamber may fill the reservoir pocket (similar tofilling the barrel of a syringe) during the “withdraw” state of the“syringe pump”, and flow into the second purification chamber during the“dispense” state of the “syringe pump.” The size of the reservoir pocketmay vary depending on the size and function of the integrated cartridge.In certain embodiments, the reservoir has a volume of 0.01-10 ml, 0.2-5ml, 0.5-3 ml, or 1-5 ml. The shape and exact location of the reservoirmay be adjusted based on the overall design of the integrated cartridge.A check valve is installed between the nucleic acid waste chamber andthe second purification chamber to ensure unidirectional flow of theliquid. The washin step in the second purification chamber can beaccomplished by closing the cartridge valve, open vent (not shown) inthe nucleic acid waste chamber and continuously flow the protein washingbuffer from the buffer reservoir into the second purification chamberthrough the “syringe pump.”

In yet another embodiment, the second purification chamber is attachedto a gear pump that continuously recirculates solution through the fritin the second purification chamber (FIG. 3C). The gear pump can operatebi-directionally and no check valve is needed between the nucleic acidwaste chamber and the second purification chamber. The protein washingstep may be accomplished by closing the cartridge valve, opening thevent in the nucleic acid waste chamber and continuously flowing theprotein wash buffer from the buffer reservoir into the secondpurification chamber through the gear pump.

The Microarray-Based Sample Analysis (MBSA) System

Also disclosed is a microarray-based sample analysis (MBSA) system. Asshown in FIG. 4, an embodiment of the MBSA system 200 includes acartridge holder 210, a fluid control subsystem 220, an opticalsubsystem 230, control software 240 and, optionally, a thei inocycler250.

The cartridge holder 210 is configured to be connected to an integratedcartridge 100 through a fluidic manifold 212. The cartridge holder 210also holds the integrated cartridge 100 in a position to facilitateinteraction between various components of the integrated cartridge andthe subsystems of the MBSA system. For example, a properly attachedcartridge would position the PCR chamber 5 of the cartridge between theheating elements of the thermocycler 250 and allow optical interrogationof the microarray 4 by the optical subsystem.

The fluid control subsystem 220 controls the fluid movement within theMBSA system 200. The subsystem includes multiple fluid containers,pumps, valves and tubings. The fluid control subsystem 220 is directlyconnected to the integrated cartridge 100 through the fluidic manifold212

The optical subsystem 230 is designed to capture images of themicroarray 4 of the sample analysis unit 2 after hybridization to asample. In certain embodiments, the optical subsystem is specificallydesigned for low-level fluorescence detection on microarrays. In oneembodiment, the optical subsystem uses confocal or quasi-confocal laserscanners that acquire the microarray image pixel by pixel in the processof interrogating the object plane with a tightly focused laser beam. Thelaser scanners offer the advantages of spatially uniform sensitivity,wide dynamic range, and efficient rejection of the out-of-focus straylight. The laser scanners, however, are very delicate and expensivedevices.

In another embodiment, the optical subsystem uses imaging devices withflood illumination, in which all the microarray elements (features) areilluminated simultaneously, and a multi-element light detector, such asa CCD camera, acquires the image of microarray either all at once or ina sequence of a few partial frames that are subsequently stitchedtogether. Compared to laser scanners, CCD-based imaging devices havesimpler designs and lower cost. CCD-based imaging systems are anattractive option for both stand-alone and built-in readers incost-sensitive applications relying on microarrays of moderatecomplexity (i.e., having a few hundred or fewer array elements).Commercial instruments typically use cooled CCD cameras and employexpensive custom-designed objective lenses with an enhancedlight-collection capability that helps to balance, to some extent, thelow efficiency of the excitation scheme.

In another embodiment, the optical subsystem contains an imaging devicethat uses a non-cooled CCD camera. Although non-cooled cameras typicallyhave a noticeably higher dark current as compared to the cooled models,the optical subsystem could provide the required sensitivity withoutusing exposures in excess of a few seconds by (1) increasing theexcitation intensity, or (2) employing an objective lens with high lightcollection efficiency; or (3) using the above two approaches incombination. The light source can be a conventional light source, suchas a metal halide or mercury bulb, a laser-based system, or ahigh-intensity LED.

In another embodiment, the optical subsystem has afluorescence-independent imaging (FII) mode as a supplementary imagingmode of microarray reader operation. The FII mode allows imaging thearray elements regardless of their fluorescence level.

The practical implementation of FII is technically challenging in bothmicroarray scanners and imagers using flood illumination. The problem isespecially difficult when the microarrays to be imaged are themainstream planar arrays, because the layer of biomolecular probesimmobilized on the microarray substrate is too thin to produce anoticeable change in the intensity of light used for probing the slidesurface.

In one embodiment, the present invention uses dark field illumination inreflected light for imaging gel arrays printed on opaque (black) plasticsubstrates. In another embodiment, the present invention uses obliqueillumination in transmitted light for imaging gel arrays printed ontransparent (glass) slides. In both cases, the light source used for FIIcould be any light source emitting within the transmission band of thereader's emission filter.

The control software 240 is designed to control all the components ofthe MBSA system, including the fluid control subsystem 220, the opticalsubsystem 230, the thermocycler 250 and any additional subsystems. Thecontrol software 240 may be installed on any computer that is connectedto the MBSA system and provides a user interface for the MBSA system. Inone embodiment, the MBSA system includes a controller that contains aprocessor, memory, and a user interface

The thermocycler 250 is configured to provide heating and cooling to thePCR chamber 5 of the integrated cartridge 100. In one embodiment, thethermocycler 250 is a bladder thermocycler described in U.S. patentapplication Ser. Nos. 11/843,843 and 12/232,669, both of which areherein incorporated by reference in their entirety.

In another embodiment, the MBSA system further includes an isothermalheating system for the microarray chamber 3. In one embodiment, theisothermal heating system contains an air heating unit and a blower thatblows heated air through a nozzle to the back side of the microarraychamber. Thermal control is achieved with a thermocouple at the nozzleor near the microarray chamber.

In another embodiment, the microarray chamber is heated by a singlesided bladder so that the microarray chamber can be imaged with theoptical system from the other side. This embodiment would allowreal-time PCR monitoring of the gel-elements in the array as describedelsewhere (Khodakov et al., BioTechniques, (2008) 44:241-248).

In another embodiment, the MBSA system further includes a cell lysissubsystem. In one embodiment, the cell lysis subsystem includes amagnetic stirrer that produces a rotating magnetic field. In anotherembodiment, the cell lysis subsystem produces sonication or vibration tothe integrated cartridge to facilitate cell lysis.

EXAMPLES Example 1 Components of the Microarray Based Sample Analysis(MBSA) System A. The Fluidic Subsystem

The fluidic subsystem consists of three types of functional components:bidirectional microfluidic pumps, selection valves, and cartridge “pin”valves. FIG. 5 is a schematic of the fluidic subsystem showing pumps andselection valves connected to the integrated cartridge. The fluidiclayout uses a combination of bidirectional pumps and selection valves,available from Global FIA. As shown in FIG. 5, the fluidic subsystem isconnected to the integrated cartridge through a fluidic manifold 90 andkeeps the various reagents in storage containers outside the integratedcartridge 100.

FIG. 6A shows an embodiment of a fluidic manifold 90. In thisembodiment, the manifold 90 allows two fluidic fittings 91 to beconnected for each cartridge port 72. The manifold has eight bosses 94with luer tapers. These bosses have a fluidic channel that provides aflow path from the fluidic fittings to the cartridge port. In thisembodiment, the fluidic manifold 90 contains a holder 96 that isconfigured to hold a pair of thermocycler bladders, such as thosedescribed in U.S. patent application Ser. Nos. 11/843,843 and12/232,669, in such a manner so that the amplification chamber 5 of thesample analysis unit 2 of the integrated cartridge 100 is positionedbetween the two thermocycler bladders 92 when the integrated cartridge100 resides on the fluidic manifold 90. FIG. 6B shows a fluidic manifold90 with the integrated cartridge 100 and the thermocycler bladders 92.

Liquid flow within the integrated cartridge 100 is also controlled byon-board cartridge valves 80. FIG. 7 shows the location of the on-boardmicrofluidic cartridge valves 80 in an embodiment of integratedcartridge 100. FIG. 8 shows an embodiment of a valve 80.

In this embodiment, the valve 80 is made of a machined plastic body 82,a spring 84, an inner o-ring 86 and an outer o-ring 88, and are screwedin place. The machined parts can also be injection molded to reducecost. In another embodiment, the valves further contain a valvealignment fixture (not shown) for rapid and consistent insertion on thecartridge. The valves can be secured on the cartridge by laser welding,heat staking, ultrasonic welding or snap fitting in lieu of beingscrewed in place.

The on-board valves 80 may be opened and closed by manually turningscrews to push the valve shaft in and out. Alternatively, an automatedlinear actuator panel with supporting electronics and software may beimplemented to control the on-board cartridge valves electronicallybased on the required protocol.

In one embodiment, the fluidic subsystem includes two bidirectionalpumps: one for sample preparation and one for polymerase chain reaction(PCR) and/or APEX. These pumps connect to a carrier solution and thecenter of a selection valve. The pumps have two types of holding coilsassociated with them: a circular coil to provide mixing by the“racetrack” effect and a switchback coil that alternates betweenstreamlines that undergo the racetrack effect.

In this embodiment, the fluidic subsystem contains three 10-portselection valves to provide flexibility and to isolate samplepreparation from the PCR/APEX fluidics. One of the selection valvesserves to aliquot sample preparation reagents to the cartridge with anembedded Akonni TruTip, another serves to aliquot PCR and APEX pelletrehydration buffers to the cartridge, and the third serves as a means ofventing or applying positive pressure to reservoirs on the cartridge.Increasing the number of ports per selection valve is one way toincrease the number of cartridges per instrument without adding hardwarecomplexity.

The cartridge pin valves serve two purposes: (1) they allow a singlereagent supply line to serve multiple reservoirs on the cartridge and(2) they control liquid movement that would otherwise be directed inunwanted pathways due to the compliance of air. Miniature linearactuators open and close the cartridge pin valves with forces less than35N.

Small holders were designed and implemented to rigidly position theactuators concentric with the shaft of the pin valves. The holder designallows the actuator to be rotated along the axis of the pin valve shaftto tightly pack multiple actuators into place. This design is necessarybecause the body of the actuator protrudes from the axis, giving it aquasi-elliptical profile (i.e., the actuators are not axisymmetric). Theactuators are secured to an I-Beam to rigidly secure them to theinstrument. The arm of the actuators penetrate through holes on a secondI-Beam, which provides support for the cartridge. This second I-Beam hasa linear pattern of threaded holes that allow standoffs to be fastenedto it. The standoff also has a threaded hole that accepts a bolt, whichpenetrates through the cartridge. The bolts keep the cartridge in afixed position with respect to x, y and z dimensions. In one embodiment,the pin valves are secured by screws. In another embodiment, multiplepin valves are being actuated by a single actuator.

B. The Bladder Thermocycler Subsystem

The bladder thermocycler subsystem is described in U.S. patentapplication Ser. Nos. 11/843,843 and 12/232,669, both of which areincorporated herein by reference in their entirety.

In one embodiment, the bladder thermocycler subsystem consists of 2pumps, 3 three-way valves, three heaters (two for the denaturing flowloop and one for the annealing/extension flow loop), 2 reservoirs thatserve as bubble traps and refilling access, a radiator, and a bladderassembly having two flexible bladders facing each other. In thisembodiment, the pump is a diaphragm pump with a flow rate range of0.15-2.00 liters per min, and operates up to 82° C. To protect thetemperature of the pump from the heaters, a PVC fitting was used toinsulate the heater from the pump.

The ramp times with this system are approximately 10° C./sec for bothheating and cooling. For preliminary evaluation of performance for thisiteration of the bladder thermocycler, a PCR reaction was performed in aCepheid Smart Cycler tube (flat reaction tube) inserted between thebladders in the bladder assembly of the bladder thermocycler and in astandard 0.2 ml PCR tube inserted in an MJ Research thermocycler. Boththermocyclers generated equivalent levels of product. FIG. 9 showsanembodiment of a detachable sample analysis unit 2. In this embodiment,the detachable sample analysis unit 2 is a flow cell having an arraychamber 22 with a microarray 23, an inlet port 24 and an outlet port 25,and a PCR chamber 26 with an inlet port 27 and an outlet port 28. Asshown in FIG. 6B, the flow cell is attached to one side of theintegrated cartridge so that the PCR chamber is extended from the sideof the integrated cartridge into the bladder heating units of thethermocycler which is attached to the fluidic manifold. FIG. 10demonstrates PCR in a flow cell and subsequent APEX using the bladderthermocycler, showing equivalent discrimination in both a tube-basedformat and a Cepheid Smart Cycler tube on the bladder thermocycler.

In one embodiment, the MSTA system is designed for room temperaturehybridizations. In another embodiment, the MSTA system comprises anisothermal heating system for heating the array chamber. The isothermalcontrol can be achieved by blowing heated air at the back side of thearray chamber with a thermocouple at the nozzle of the air flow tube. Incertain embodiments, the isothermal heating system is capable ofmaintaining the temperature of the array chamber in the ranges of 20°C.-65° C., 20° C.-75° C., 20° C-85° C. or 20° C-95° C.

C. The Optical Subsystem

FIG. 11A is a schematic of an embodiment of an optical subsystem. Thelow-cost optical subsystem is developed for reading arrays of arelatively low complexity (number of array elements <50). The CCD cameraused in this subsystem of the reader is a miniature non-cooled camera of⅓″ optical format with resolution 659×493 pxl and a pixel size of7.4×7.4 μm (e.g. a Prosilica model EC650).

Both the objective and camera lenses are identical F/1.5 single-elementaspheric lenses such as Edmund Optics part #NT47-732. The lenses have aneffective focal length of 37.5 mm. Since the magnification of theimaging system is equal to 1, the pixel size in the object plane is thesame as the pixel size of the CCD sensor (7.4 μm).

The useful field of view (FOV) is about 2 mm in diameter, which isenough for imaging arrays comprising up to 50 spots under assumptionthat the array pitch is 300 μm. The FOV-limiting factor is the fieldcurvature, which predictably is relatively high for this simplisticoptical setup.

Another distinctive feature of the optical design (in addition to theobjective lens with high light-collection efficiency ensured by the lowf-number of 1.5) is the co-axial illumination scheme that employs a 15mW green (532 nm) DPSS laser and a multimode optical fiber that is usednot only for beam delivery, but also for beam shaping and specklesuppression.

Because of the multimode nature of the fiber, the Gaussian intensitydistribution in the laser beam at the fiber input face is transformedinto a top-hat-like distribution at the fiber output. The collimatingand beam-focusing lenses work together to project the image of the fiberend face onto the object plane of the objective lens. As a result, theintensity distribution in the object plane is similar to that at thefiber output, whereas the size of the illuminated spot is determined bythe fiber core diameter and the magnification factor of the two-lensprojection system.

The speckle suppression is achieved with the help of a miniaturevibrating motor attached to the fiber (see FIG. 11A). Vibrating thefiber results in rapid modulation of the phase shifts between differentfiber modes, which translates into high-frequency intensity oscillationsin the speckle pattern. The latter are effectively smoothed out in theprocess of taking the image even for exposures as short as 100 ms.

In one embodiment, a low-cost optical subsystem scans a microarray andstitches the images together to generate a complete picture of themicroarray. In another embodiment, arrays on multiple cartridges areconsecutively scanned using a linear motion system.

FIG. 11B shows a typical image of a Cy3 array with a pitch of 300 μmtaken with the optical subsystem described in FIG. 11A with the co-axialillumination.

FIG. 12A is a diagram of another optical subsystem. This version ofoptical subsystem was developed for imaging arrays with the number offeatures up to 200, which is sufficient for many diagnostic assays.Expanding the field of view calls for the objective lens with a highdegree of aberration correction. It also makes it necessary to use alens with a higher f-number. An example of an objective that satisfiesthe above requirements is a Leica's infinity-corrected Planapo 2× lenswith a working distance of 39 mm and a numerical aperture of 0.234. Itshould be noted that the objective and camera lenses in the imagingoptical path of the optical subsystem are shown as single-element lensesfor the sake of simplicity only. In fact, both these lenses are highlycorrected multi-element optics that work at infinity conjugates and havethe focal length equal to each other. So the magnification factor of theentire lens system is 1.

In another embodiment, the focal length of the beam-focusing lens wasincreased to expand the illuminated spot in the object plane to about 8mm. It was found experimentally that with the spot diameter that large,the non-uniformity of illumination caused by the oblique angle of beamincidence did not exceed 5%. FIG. 12B represents a typical image of am11×18 Cy3 array taken with the optical subsystem employing the obliqueillumination scheme (FIG. 12A). As described above, the array pitch is300 μm, and a characteristic diameter of the array spots is about 120μm.

FIG. 13A shows another embodiment of the optical subsystem. In thisembodiment, the laser in the oblique illumination design is replacedwith a high-brightness light-emitting diode (LED). The LED illuminatorshown implements a Köhler illumination scheme (A. V. Arecchi, T.Messadi, and R. J. Koshel, Field Guide to Illumination, SPIE Press Book,Vol. FG11, Aug. 31, 2007, ISBN: 9780819467683, which is incorporatedherein by reference) for projection systems and includes a clean-upfilter placed between the collector and condenser lenses and abeam-steering mirror directing the excitation light at the object planeat oblique angle. The filter is intended for rejecting the spectralcomponents of LED light overlapping with the emission spectrum of thedye used for DNA labeling (e.g. Cy3). Although the embodiment shown inFIG. 13A is a preferred one, it will be apparent to those skilled in theart that the beam delivery system with a mirror may be modified withoutdeparting from the scope and spirit of the invention. In particular, aliquid light guide or fiber optic bundle could be introduced in theoptical train to facilitate beam delivery from a remote light source.

In other embodiments, the optical subsystem comprises a miniaturelow-power LED for FII. FIG. 13B shows an embodiment of an optical traincapable of both the fluorescence imaging and FIT using obliqueillumination in transmitted light. FIG. 13C shows an embodiment of anoptical train capable of both the fluorescence imaging and FII usingdarkfield illumination in reflected light. The optical train in FIG. 13Crequires less space behind the slide holder (i.e., the object plane).FIG. 13D shows an exemplary technical implementation of a collimatedsource. The light emitter used is a low-intensity yellow LED with a peakemission wavelength of 591 nm.

FIG. 14A shows an exemplary image of Akonni MRSA assay slide recordedusing oblique illumination with a collimated beam of LED light source.FIG. 14B shows the normalized pixel intensity profile of an arrayelement in FIG. 14A. Although images with the highest contrast weretypically obtained using oblique illumination with a collimated lightsource, other optical arrangements for oblique illumination are alsopossible. The general requirement for such schemes is asymmetricaldistribution of illumination intensity between different azimuths. TheFII array image can be processed for the purposes of gridding and spotmorphology analysis using relatively simple image processing tools, suchas the image processing tools in the ImageJ software(http://rsbweb.nih.gov/ij/). FIG. 15 is a composite of images showing agel array image processed by different methods available in the ImageJsoftware. FIG. 16 is a composite showing evaluation of spot morphologyusing the ImageJ software.

FIGS. 17A and 17B are graphs showing evaluation of morphology for thegel spots in panel A of FIG. 15. In this study, FII imaging usingoblique illumination with a collimated beam allowed both detection ofarray spot centers and identification of damaged spots with considerablemorphology abnormalities. The data suggest that, for the bestsensitivity of morphology analysis, the spots need to be characterizedby a combination of parameters. For example, by spot area and parametersof best fitting ellipse;

It should be noted, however, that array image QC cannot rely solely onthe FII imaging mode: some of the image features that may seem innocuousin an FII image can, in fact, be a source of considerable interferencefor spot fluorescence intensity measurements (e.g., the particle markedby the arrow in panels A and D of FIG. 16).

Example 2 Detection of Bacteria in Test Samples with IntegratedCartridge and the Microarray Based Sample Analysis System

The sequence of events for the MBSA system are as follows. The userintroduces a sample into the integrated cartridge. The system thenperforms the following automated steps: prepare the samples, perform PCRusing a Bladder Thermocycler™, mix the PCR product with a hybridizationand/or APEX reaction mixture, transfer the mixture to the microarraychamber, perform hybridization to the microarray, and detect themicroarray image.

In one embodiment, the sequence of steps for preparing samples on thecartridge are: introduce the sample into the sample tower, introduce thebind buffer into the sample tower, mix with air, transport the samplemix to the TruTip tower, toggle between the towers, dispense to waste,introduce the wash buffer to the TruTip tower, toggle between thetowers, dispense to waste, introduce the elution buffer, and dispense tothe elution tower.

The sequence of steps for PCR are: add PCR mix to the elution tower orreconstitute a lyophilized reagent pellet with elution buffer followingsample processing, dispense to PCR chamber on flow cell, close allcartridge pin valves, and start thermocycling.

The sequence of steps for APEX are: flush PCR chamber with APEX bufferand add to APEX reservoir, thoroughly mix, add APEX reagent to arraychamber, incubate at 65° C., wash, and image.

In one experiment, 500 μL of 10⁵cfu/mL of Streptococcus pyogenes, whichhad been lysed offline by vortexing for 2 minutes, was introduced intothe sample chamber of an integrated cartridge. The cartridge wasconnected to a prototype MBSA system comprising a cartridge adaptor, afluidic control subsystem for sample preparation and processing, athermal control subsystem for PCR amplification, an optical subsystemand a data acquisition board and PC for image acquisition.

The following steps are controlled by the analytical system. Bind bufferwas added to the sample mixing chamber, and mixed with the sample by airbubbles that migrate from the bottom of the chamber to the top of thechamber. The air bubbles were controlled by regulated air flow from theinstrument pump. The mixed sample was then introduced into the samplepurification chamber, which contains the silica extraction filter thatbinds specifically to DNA, and cycled 5 times back and forth across thematrix to improve binding efficiency. The unbound material was thendirected to the waste chamber in the cartridge by means of on-boardcartridge valves. Leaving the waste in the cartridge preventscontamination with subsequent samples. A wash buffer was subsequentlytoggled 5 times between the TruTip tower and the sample preparationtower and then directed to the waste chamber. One hundred and twentymicroliters of elution volume was then introduced from the analyticalsystem onto the cartridge through the TruTip tower and then directedinto the elution chamber, which removes the bubbles.

A 10 ul aliquot of the eluted DNA was removed and processed on a Roche480 real-time PCR Light Cycler. A second aliquot of the eluted DNA wasalso removed from the elution chamber and amplified “off-line” in arepresentative flow cell. A PCR master mix is then introduced into theelution chamber. The eluted sample was mixed with the master mix bybubbling air from the bottom of the elution chamber. The mixed samplewas then directed, via on-board valves into the PCR chamber of a flowcell attached to the integrated cartridge, which was positioned betweentwo compliant bladders of the thermocycler when the cartridge isinserted in the fluidic docking station. A manufacturing benefit to theuse of compliant bladders is that because they expand and make intimatecontact with the cartridge flow cell, they can accommodate a large rangeof flow cell thicknesses and positional variations without changing thedesign. Forty cycles of two-temperature PCR was performed by oscillatingtwo temperature fluids through the bladder using the BladderThermocycler™ method. This method is based on flowing heated liquidsinto a flexible membrane (“bladder”) that makes intimate contact withthe PCR chamber. Valves switch between thermally-controlled reservoirsto provide a rapid change in temperature. This thermocycling approachresults in PCR protocols that are approximately 4× faster thanconventional slide thermocyclers, which rely on Peltiers for heating andcooling.

Following amplification, the PCR product was automatically transferredto the microarray chamber of the flow cell through the on-boardcartridge valves and hybridized to the microarray in the microarraychamber for one hour at room temperature. The microarray was then readwith an Aurora PortArray 5000 imager. FIG. 18A shows an image of thehybridized microarray taken from the Aurora Port Array 5000. Thepositive signals are indicated by the boxes.

The second aliquot of the eluted DNA was amplified “off-line” in a flowcell identical to the flow cell attached to the integrated cartridge.Briefly, the DNA sample was manually loaded into the PCR chamber of theflow cell and amplified as described above. The amplification productwas then manually loaded into the microarray chamber and hybridized tothe microarray in the microarray chamber for one hour at roomtemperature. The microarray was then read with an Akonni imagerspecifically designed for gel element arrays and flow cell assemblies.FIG. 18B shows an image of the hybridized microarray taken from theAkonni imager. The positive signals are indicated by the boxes.

In another experiment, 500 μl Bacillus anthracis sample (10 cfu /ml inwater), was processed and analyzed using the procedure described above.FIG. 19A shows an image of the microarray that indicates positivedetection of Bacillus anthracis in the test sample. FIG. 19B is thearray map for the microarray used in FIG. 19A.

In another experiment, 500 μl Streptococcus pyogenes sample (10⁴ cfu/mlin water) was processed using an integrated cartridge with a magneticbead mixer in the sample preparation chamber. Glass beads were added tothe chamber and the bead mixer was agitated to lyse the organism in thechamber. FIG. 20 shows PCR results of DNA extracted with the mixer. Thevortexing method was accomplished by mixing beads to sample volume at aratio of 50/50 and vortexed for 2 minutes in a 1.5 mL micro centrifugetube.

Example 3 Genotyping of Single-Nucleotide Polymorphism (SNP Typing) withIntegrated Cartridge and the Microarray Based Sample Analysis System

This example demonstrates the feasibility of a microfluidic-controlledsystem for SNP-typing of physical appearance markers that could beutilized for forensic applications. The experimental setting combinesthe components for sample preparation, PCR amplification andAllele-specific Arrayed Primer Extension (AS-APEX) to form an integratedsystem to detect markers for eye color. The system contains a liquidhandling sub-system (e.g., pumps), thermal cycling sub-system (e.g.,Akonni Bladder Thermocycler), optical instrument (e.g., Akonni Reader),a disposable, integrated cartridge (i.e., Akonni TruTip, AkonniPCR/TruArray flow cell, microfluidic circuits, and microfluidic valves)and a cartridge docking station.

The six SNPs listed in Table 1 are used as test models for theexperiment. These SNPs have been reported in the literature as majorindicators of eye color.

TABLE 1 Six SNPs Identified as Major Determinants for Eye Color CommonMinor Allele/ Allele/ SNP-ID Chr Position Gene Eye color Eye color Notesrs12913832 15 26039213 HERC2 G/Blue A/Brown rs1800407 15 25903913 OCA2C/Brown T/Blue T/associated with Green/Hazel rs12896399 14 91843416SLC24A4 T/Blue G/Brown rs16891982 5 33987450 SLC45A2 G/Blue C/BrownC/associated with Black hair rs1393350 11 88650694 TYR G/Brown A/Bluers12203592 6 341321 IRF4 C/Brown T/Blue

Sequence information for the SNPs was compiled and forward and reversePCR primers were synthesized. Single-plex PCR reactions for eachamplicon were tested and optimized. Systematic pooling of the primers ina multiplex format to produce a standard 6-plex PCR reaction for the eyecolor amplicons was achieved. This standard 6-plex PCR reaction wasconverted to a low temperature (LowTemp) 6-plex PCR reaction. LowTempPCR involves using a chemical to supplement temperature annealingstringency. By adding formamide (a common PCR additive) to the mastermix, the denaturing temperature was reduced to 85° C. instead of thenormal 95° C. This strategy allows more choices of plastics andadhesives for our integrated cartridge; thereby lowering manufacturingcosts. In addition, the lower temperature allows more material choicesfor the bladder thermocycler components and plumbing. In one embodiment,a single enzyme is used for both PCR and APEX.

FIG. 21A displays a microarray image and fluorescent intensities of gelspot signals for each amplicon generated in the LowTemp 6-plex PCR. Allsix amplicons were detected, with amplicon signal intensities exceedingthe acceptable threshold for a positive call on the array. Whilerelative signal intensities vary amongst the products, the signalintensities can be fine tuned by adjusting primer concentrations in themultiplex PCR and/or length of primers used for APEX.

In the APEX approach, primers are designed so that a single primer isimmobilized within each gel element on the array such that the ending 3′base is at the SNP site. A separate primer is designed for each SNP tobe detected. For instance, if there is a possibility for an A or a C ata certain SNP site, then a separate primer is designed for each, oneending in a 3′ A and one in a 3′ C. Extension by polymerase is inhibitedif the 3′ nucleotide of the primer is mismatched to the target. In thepresence of the correct target and matched 3′ base, polymeraseincorporates fluorescently labeled nucleotides to produce the finalsignal.

In one experiment, genomic DNA was purified from 50 ul blood sampleusing the integrated cartridge. In this process, all sample and samplewaste along with all reagents/buffers that came in contact with thesample were maintained on the cartridge. Used reagents/buffers weremoved and stored in the cartridge waste chamber. The entire process wasperformed under computer control. Real-time PCR results on extracted,purified DNA sample that was manually removed from the cartridgedisplayed a strong positive PCR signal (FIG. 21B). More importantly,this purified DNA was successfully used for manual PCR and APEX to callthe correct genotype (Table 2). Briefly, automated, integratedcartridge-purified DNA was used for (manual PCR (20 ng DNA)/APEX typing.The fluorescent intensities of each primer on the array were imaged, andraw signal intensity data was used to generate primer (allele) signalratio values for each SNP. The called genotype matched the genomic DNAsequencing results.

TABLE 2 Genotyping of blood samples using primer (allele) signal ratiovalues Blood DNA Ratios Sample Grand ID Description Average AverageAverage Results 50 Rs1393350: G/A 0.75 0.77 0.76 A/G 53 Rs1393350: A/G1.33 1.30 1.32 23 Rs16891982: C/G 5.33 5.48 5.40 C 24 Rs16891982: G/C0.19 0.18 0.19 77 Rs1800407: G/A 5.11 5.13 5.12 G 78 Rs1800407: A/G 0.200.20 0.20 33 Rs12913832: A/G 3.62 3.64 3.63 A 34 Rs12913832: G/A 0.280.27 0.28 66 Rs12896399: G/T 0.34 0.62 0.48 T 69 Rs12896399: T/G 2.911.62 2.26 71 Rs12203592: C/T 31.55 10.83 21.19 C 73 Rs12203592: T/C 0.030.09 0.06

Example 4 Software Development: DX3000 Automated Task Execution Program

A software program, designated DX3000 Automated Task Execution Programwas created to control the integrated assay system. Code was written inLabview on a National Instruments industrial computer (NI 3110) tocontrol the three major sub-systems (Fluidic Handling Sub-System,Thermocycler Sub-System, and Optical Sub-System). The NI 3110 has adual-core processor where one core executes tasks for Windows and theother core executes tasks for a Real-Time operating system (OS). Thisarchitecture allows for the execution of high level Windows tasks suchas managing the user-interface, serial communication and imageprocessing, as well as low-level deterministic tasks such as control ofthe heaters, linear actuators, thermocycler pumps, and precisely timedevents. The Real-Time OS communicates with the Ethercat I/O module,which scans analog input, analog output, and digital I/O modules.

Each of the three major sub-systems utilize resources from both theWindows and Real-Time OS and require communication between the twooperating systems. Within the Windows environment a sequence of tasks,created by the user, is managed and communicated to the appropriateprocess on either the Real-Time OS or the Windows OS via sharedvariables. Tasks associated with the Fluidic Handling Sub-Systeminclude: change position of the selection valve(s), close/open cartridgevalve(s), and dispense/aspirate with microfluidic pump. Tasks associatedwith the Thermocycler Sub-System include: warm up the thermocycler andinitiate thermocycling. And tasks associated with the Optical Sub-Systeminclude: initiate APEX heating and acquire an image. Sequences can besaved and imported into the DX3000 Automated Task Execution Program.

The components, classified as being controlled by the Fluidic HandlingSub-System, include: 2 bi-directional pumps, 3 selection valves, and 8linear actuators, which open and close the cartridge valves. Thebi-directional pumps and the selection valves are controlled by a USBserial port. Serial commands are communicated to them via Labviewdrivers, developed by Global FIA. The control commands for the pump aredirection, flow rate, volume and address, and the control commands forthe selection valve include selection valve port number and address.Whereas these commands are executed entirely within Windows, the linearactuators are controlled primarily from the Real-Time Operating System.Linear actuators, available from Firgelli Technologies Inc. (Victoria,Canada) are controlled by an H bridge of 4 MOSFETs for each valve. Twodigital I/O lines per actuator trigger the actuator to move forward,move backward, or remain at rest. When the actuator is at rest, no poweris required. The actuators include an internal potentiometer to providefeedback of the position of the actuator arm. A calibration routine isused to determine the extents to which the actuator arm reaches, whichcorrespond to a closed or an open position for the cartridge valves. Acomparison algorithm is used to compare the desired with the actuallocation of the arm. The appropriate movements are executed to reach thedesired location within a pre-determined tolerance window. Following thecalibration routine, each, all, or some combination of the cartridgevalves can be closed or opened as a task in the sequencer.

The components, classified as being controlled by the ThermocyclerSub-System, include: 2 diaphragm pumps, 3 three-way valves, 3 coiledheaters (two for the hot zone and one for the cold zone), and a fan toprovide cooling through a miniature radiator. Darlington BJT transistorssource current to the diaphragm pumps in proportion to an analog outputsignal that is controlled by the DX3000 Automated Task ExecutionProgram. Linearly proportional relays control the amplitude of an ACsignal that powers the heaters and an AC fan for the radiator. An analogvoltage output signal controls the output of these relays. Three in-linethermistors, exposed to the recirculating flow, are located after thehot zone and cold zone heaters and prior to the bladder. Two separatevirtual processes control the recirculating temperature of the hot zonefluidic loop and the cold zone fluidic loop. These virtual processesinclude an algorithm for PID control of the temperature in the loop. Theheater PID is used for a wide range of temperature control, but is slowresponding. The pump flow rate PID has a narrow range of temperaturecontrol, but is fast responding. So, both PID loops are used to achieverapid switching between the hot and cold zones with stable plateaus, asshown in FIG. 22. The radiator fan speed is typically maintained at 40%power.

The components, classified as being controlled by the OpticalSub-System, include: an LED, a camera, a heater, and an air pump. TheLED, camera and air pump are turned on and off by solid state relays. Apulse width modulation algorithm controls the duty cycle of AC power tothe heater using a solid state relay. The air pump flow rate ismaintained at a constant rate. A thermocouple, internal to the heater,provides feedback to a PID virtual interface.

Example 5 Protein and Nucleic Acid Purification on the Same Cartridge

The integrated cartridge can be modified to contain both protein andnucleic acid purification capabilities. FIG. 23 shows an embodiment of adual-function integrated cartridge 300. Briefly, the cartridge 300contains a protein purification module and a nucleic acid purificationmodule. The protein purification module includes a protein waste towerT1, a protein purification tower T2, a protein elution tower T3,cartridge pin valves CV1, CV2, CV3 and CV4 and Luer-activated valvesLV1, LV2, LV3 and LV4. The nucleic acid purification module includes anucleic acid waste tower T4, a sample preparation tower T5, a nucleicacid purification tower T6, a nucleic acid elution tower T7, cartridgepin valves CV5, CV6, CV7, CV8 and CV9, and Luer-activated valves LV5,LV6, LV7, LV8 and LV9.

In an embodiment, nucleic acid is purified via the following procedure:

1) A. Close all CVs

-   -   B. Add 500 μL sample to T5 through sample inlet    -   C. Open CV6 and CV7    -   D. Add 500 μL cell lysis solution to T5 through LV5 (with LV7        venting to atmosphere)

2) Add air for mixing to T5 through. LV5 (with LV7 venting toatmosphere)

3) Incubate for 5 minutes at room temperature

4) A. Close CV6, Open CV8 (CV7 remains open, CV8 will stay open untilthe start of Protein Purification protocol)

-   -   B. Flow air through LV7 (with LV8 venting to atmosphere)    -   C. Flow air through LV8 (with LV7 venting to atmosphere)    -   Repeat Steps B & C five times. Mixture will flow back and forth        from T5 to T6, ending in T6

5) A. Close CV7, Open CV2

-   -   B. Flow air through LV8 (with LV2 venting to atmosphere) to push        1 mL of mixture into Ti

6) A. Open CV6, Close CV2

-   -   B. Add wash to T6 through LV5 (with LV8 venting to atmosphere)    -   C. Close CV6, Open CV7    -   D. Flow air through LV8 (with LV7 venting to atmosphere)    -   E. Flow air through LV7 (with LV8 venting to atmosphere)    -   Repeat Steps D & E five times. Wash will flow back and forth        from T5 to T6, ending in T6    -   E. Close CV7, Open CV5    -   F. Flow air through LV8 (with LV6 venting to atmosphere) to push        wash into T4

7) A. Open CV6, Close CV5

-   -   B. Add air to T6 through LV5 (with LV8 venting to atmosphere)

8) A. Add elution to T6 through LV5 (with LV8 venting to atmosphere)

-   -   B. Close CV6, Open CV9    -   C. Flow air through LV8 (with LV9 venting to atmosphere)    -   D. Flow air through LV9 (with LV8 venting to atmosphere)    -   Repeat Steps C & D five times. Elution will flow back and forth        from T6 to T7, ending in T7

The proteins in the 1 ml sample mixture stored in T1 (see step 5B) ispurified via the following procedure:

9) A. Open CV1 and CV2, close all other CVs

-   -   B. Add protein binding solution to T1 through LV1 (with LV2        venting to atmosphere)

10) Add air for mixing to T1 through LV1 (with LV2 venting toatmosphere)

11) Incubate for 2 minutes at room temperature

12) A. Open CV3, Close CV1 (CV2 remains open, CV3 will stay open untilthe end of the protocol)

-   -   B. Pull pump arm backwards to pull 250 μL of solution from T1        through T2 via the connecting channel (with LV2 & LV3 not        venting)    -   C. Push pump arm forward to push 250 μL of solution from T1        through T2 via the connecting channel (with LV2 & LV3 not        venting)    -   Repeat Steps B & C ten to fifteen times. Solution will flow in a        single direction loop due to one-way check valve on cartridge,        which is located directly above    -   CV2 and directly below T1.    -   D. Open CVS, Close CV2    -   E. Flow air through LV3 to push solution into T4 (with LV6        venting to atmosphere)    -   Note: Volume of Protein Waste Chamber (T1) is now full, so        Nucleic Acid Waste Chamber is being used (T4)    -   F. Open CV1, Close CV5

13) A. Add wash to T2 through LV1 (with LV3 venting to atmosphere)

-   -   B. Open CV5, Close CV1    -   C. Flow air through LV3 to push wash into T4 (with LV6 venting        to atmosphere)    -   D. Open CV1, Close CV5    -   Repeat Steps A - D five times. Wash will be added to T2 and then        flow into T4

14) Add air to T2 through LV1 (with LV3 venting to atmosphere)

15) Increase flow rate of air to blow out residual liquid

16) A. Add elution to T2 through LV1 (with LV3 venting to atmosphere)

-   -   B. Close CV1, Open CV4    -   C. Flow air through LV3 (with LV4 venting to atmosphere)    -   D. Flow air through LV4 (with LV3 venting to atmosphere)    -   Repeat Steps C & D five times. Elution will flow back and forth        from T2 to T3, ending in T3

In one embodiment, the sample lysis solution is 6M guanidineisothiocyanate. In another embodiment, the protein binding solution is0.1% trifluoroacetic acid.

FIG. 24 shows a prototype MBSA system 500 that include an integratedcartridge 510 with a sample analysis unit 2 (i.e., a flow cell with anarray chamber and a PCR chamber), a fluid control subsystem 520, anoptical subsystem 530, a bladder thermocycler subsystem 550, and afluidic manifold 560. In this embodiment, the fluid control subsystem520 contains a setup capable of parallel processing of protein andnucleic acid in a dual-function integrated cartridge. The setup includestwo pumps 521 and 523, and three selection valves 525, 527 and 529. Onepump and one selection valve would be for nucleic acid extraction andthe other pump and another selection valve for protein. The thirdselection valve would be used to open and close the vents. Such asetting would allow parallel processing of protein and nucleic acidsimultaneously. The optical subsystem 530 includes a CCD camera 531,lens assembly 533 and illumination assembly 535. The bladderthermocycler subsystem 550 includes of two pumps 551 and 552, threeheaters 553, 554 and 555 (two for the denaturing flow loop and one forthe annealing/extension flow loop), two reservoirs 556 and 557 thatserve as bubble traps and refilling access, and a bladder assembly 558having two flexible bladders facing each other.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding of theprinciples of construction and operation of the invention. Suchreferences herein to specific embodiments and details thereof are notintended to limit the scope of the claims appended hereto. It will beapparent to those skilled in the art that modifications may be made inthe embodiment chosen for illustration without departing from the spiritand scope of the invention. All the references cited in thespecification are hereby incorporated by reference in their entirety.

1.-21. (canceled)
 22. A microarray-based sample analysis (MESA) system,comprising: a cartridge holder adapted to receive a replaceablecartridge, wherein the replaceable cartridge is configured to receive adetachable, replaceable sample analysis unit containing one or morereaction chambers for sample analysis, wherein the one or more reactionchambers contain one or more microarrays a fluid control subsystem thatcontrols fluid flow; and an optical subsystem configured to capture animage of the one or more microarray.
 23. The MBSA. system of claim 22,further comprising: a thermocycling system that is configured to controltemperature in the reaction chamber during a reaction process.
 24. TheMBSA system of claim 23, wherein the thermocycling system comprises abladder thermocycler comprising two flexible bladders facing each other.25. The MBSA system of claim 22, wherein the optical subsystem comprisesa laser light source.
 26. The MBSA system of claim 22, wherein theoptical subsystem comprises a high-brightness LED light source.
 27. TheMBSA system of claim 22, wherein the optical subsystem is capable offluorescence imaging and fluorescence-independent imaging.
 28. The MESAsystem of claim 27, wherein the optical subsystem comprises a firstlight source for fluorescence imaging and a second light source forfluorescence-independent imaging.
 29. The MBSA system of claim 28,wherein the second light source is a light-emitting diode (LED).
 30. TheMBSA system of claim 29, wherein the LED is configured for darkfieldillumination in reflected light.
 29. The MBSA system of claim 29,wherein the LED is configured for oblique illumination in transmittedlight.
 32. The MBSA system of claim 22, wherein the one or moremicroarrays comprise a DNA microarray.
 33. The MBSA system of claim 22,wherein the one or more microarrays comprise a protein microarray. 34.The MBSA system of claim 22, further comprising: an integrated cartridgecomprising the cartridge holder and configured to receive thereplaceable sample analysis unit, the integrated cartridge furthercomprising a sample preparation chamber, a sample purification chamberin fluid communication with the sample preparation chamber and a wastechamber in fluid communication with the sample analysis unit when it isattached to the cartridge body.
 35. The MBSA system of claim 22, furthercomprising: a cell lysis subsystem, wherein the sample preparationchamber comprises sample lysis means for lysing cell samples.
 36. TheMBSA system of claim 35, wherein the sample preparation chambercomprises a sample inlet and a sample outlet, wherein the samplepurification chamber is configured to receive a first replaceable samplepurification unit comprising a housing and an extraction filter insidethe housing, wherein the extraction filter specifically finds a moleculeto be purified, and wherein the sample purification chamber has a sampleinlet that is in fluid communication with the sample outlet of thesample preparation chamber.
 37. The MB SA system of claim 22, whereinthe replaceable sample analysis unit comprises an amplification chamberfor PCR.
 38. The MBSA system of claim 22, wherein the one or morereaction chambers comprise hydrophilic flow cells, wherein the reactionchambers comprise hydrophilic interior surfaces.